CAG-Cre AAV (Serotype BR1)

Blood-brain barrier (BBB) disruption and immune cell infiltration into the central nervous system (CNS) are early features of multiple sclerosis (MS). CD8+ T cells are found in large numbers in MS lesions, and antigen (Ag) presentation at the BBB has been proposed to promote CD8+ T cell entry into the CNS. Here, researchers show that brain endothelial cells process and cross-present Ag, leading to effector CD8+ T cell differentiation. Under physiological flow in vitro, endothelial Ag presentation prevented CD8+ T cell crawling and extravasation, leading to brain endothelial cell apoptosis and BBB disruption. Brain endothelial Ag presentation in vivo was limited due to Ag uptake by CNS-resident macrophages, but still reduced the motility of Ag-specific CD8+ T cells within CNS microvessels. MHC class I-restricted Ag presentation at the BBB during neuroinflammation thus prohibits CD8+ T cell entry into the CNS and triggers CD8+ T cell-mediated focal BBB breakdown.

The AAV-BR1 vector is suitable for targeting brain microvascular endothelial cells, allowing conditional Cre-driven recombination of floxed TAP1 alleles in CNS microvascular endothelial cells of ODC-OVA TAP1^fl/fl mice to establish a BBB-specific TAP1-deficient ODC-OVA mouse model (ODC-OVA TAP1^BBB-KO). Researchers induced OT-I cell-mediated neuroinflammation in ODC-OVA TAP1^fl/fl mice (transfer of naive OT-I cells followed by infection with LCMV-OVA), or in ODC-OVA TAP1^wt/wt mice two weeks after injection of AAV-BR1-CAG-Cre as a control, or in ODC-OVA TAP1^fl/fl mice two weeks after injection of AAV-BR1-CAG-GFP as an additional control (Figure 1). Comparison of the interaction of in vitro activated CMFDA-labeled effector OT-I cells with inflamed spinal cord microvessels expressing or not TAP1 by two-photon in vivo imaging revealed that OT-I cells crawled significantly faster in the absence of TAP1 at the BBB compared with the crawling speed of mice expressing TAP-1 at the BBB (Figure 1). These data suggest that brain endothelial MHC class I-mediated Ag presentation leads to a reduction in the crawling speed of CD8+ T cells across the BBB in vivo.

Figure 1. Violin plots for crawling speed (μm/min) of in vitro activated CMFDA-labeled OT-I cells in the cervical spinal cord microvasculature lacking expression of TAP1 (TAP1 BBB-KO) or not (TAP1-BBB-Control). TAP1-BBB KO mice (green): BBB-specific TAP1 deletion was achieved in ODC-OVA//TAP1^{floxed/floxed} mice by injection of AAV-BR1-CAG-Cre viral vector two weeks before induction of autoimmune neuroinflammation. TAP1-BBB-Control (red): ODC-OVA//TAP1^WT/WT mice injected with AAV-BR1-CAG-Cre and ODC-OVA//TAP1^{floxed/floxed} mice injected with AAV-BR1-CAG-GFP. (Aydin S, et al., 2023)